PROJECT SUMMARY The aging process and many aging-associated diseases like Parkinson's disease (PD) are hypothesized to be caused by a decline in cellular energy and mitochondrial function. Cellular energy is presumably critical to many cellular processes including protein degradation, a process that is disrupted in neurodegenerative disease. Identifying genes that modulate levels of ATP, the main energy-carrying molecule in all cells, could then be critical to slowing or reversing aging-associated pathology, preserving neuron function in response to stress, and providing insight into how energy failure contributes to disease. However, that analysis has been limited by a lack of tools to screen the genome at high throughput for modifiers of ATP levels, causing a critical gap in knowledge of genetic contributors to energy failure. We have developed a unique screening paradigm to address this knowledge gap by combining genetically encoded ATP sensors with CRISPR-based whole-genome screening technology within cells exposed to acute metabolic stress. With this approach, we have identified three poorly-understood gene pathways that have a prominent impact on ATP levels specifically when cells are metabolically restricted to using only respiration. These pathways are triggered in response to cellular stress, but have also been observed in the pathophysiology of neurodegenerative diseases. While these pathways all regulate key facets of protein metabolism and stress response under normal metabolic conditions, it is unclear how compromised metabolism and low ATP affect these pathways' contributions to the elimination of protein stresses commonly associated with neurodegenerative diseases. We hypothesize that in metabolically-stressed neurons, these stress-responsive pathways exacerbate energy failure and protein accumulation. We will address this hypothesis by investigating if and how these processes affect ATP levels in neurons, as well as the functional consequences of these pathways on protein degradation and survival of metabolically stressed neurons. Successful completion of these aims will provide new insight into the relation between energy homeostasis and proteostasis, as well as the progression of neurodegeneration under metabolic stress.